To exploit the full potential of human pluripotent stem (hPS) cells for regenerative medicine, developmental biology, and drug discovery, defined, reproducible conditions for hPS cell propagation and differentiation must be identified. The long-term objective of the proposed research is to address this need by designing and implementing chemical biology strategies. The three Aims have been devised to elucidate how specific soluble and insoluble chemical cues act together to control cell fate decisions. In the past grant period, we synthesized a surface array and screened it to discover peptide-substituted surfaces that promote hPS cell self-renewal. The most effective substrates interact with glycosaminoglycans. In Aim 1, we shall investigate the role of glycosaminoglycans in hPS cell pluripotency. In Aim 2, we shall employ our surface array screen to identify insoluble signals (peptide-derived) that act in concert with soluble signals to direct hPS cell differentiation. In Am 3, we shall synthesize a series of biocompatible polymers that vary in cell binding groups and elasticity. Using these tools, we shall examine how hPS cells respond to distinct combinations of soluble, insoluble, and mechanical cues. In pursuing these Aims, we shall employ methods and ideas from organic chemistry, peptide chemistry, stem cell biology, and chemical biology.